High-density lipoprotein assay device and method

ABSTRACT

An assay device and method for measuring the concentration of HDL-associated cholesterol in a blood-fluid sample are described. The assay design is such that removal of non-HDL lipoproteins from a sample and assay of HDL cholesterol in the sample occur without interruption of the assay. The device also prevents interference by reagents used for the HDL assay with other assays carried out on the same sample.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/349,926, filed Jan. 18, 2002, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of determining theconcentration of high density lipoprotein (HDL)-associated cholesterolin a blood-fluid sample, and a diagnostic assay device for carrying outthe method.

BACKGROUND OF THE INVENTION

[0003] The amount of cholesterol present in the blood is known to berelated to the risk of coronary artery disease. Cholesterol circulatesin the blood predominantly in protein-bound form. The proteins whichtransport cholesterol are the lipoproteins, which are subdivided intothree classes based on their density. The very-low density lipoproteins(VLDL) are triglyceride-rich lipoproteins which are synthesized in theliver and ultimately converted to low-density lipoproteins (LDL), whichtransport most of the plasma cholesterol in humans. The high-densitylipoproteins (HDL) are lipoproteins which are involved in the catabolismof triglyceride-rich lipoproteins, and in the removal of cholesterolfrom peripheral tissues and transport to the liver. An inverserelationship between serum HDL levels and risk of coronary disease hasbeen established. In particular, if the proportion of serum cholesterolassociated with HDL is low, the risk of coronary disease is increased.

[0004] In view of the importance of relative serum cholesterol levels inrisk assessment and management of atherogenic disease, considerableeffort has been spent screening large populations of both normal andhigh-risk individuals for serum levels of HDL, LDL, as well as totalcholesterol and triglycerides. The effectiveness of treatments ofhigh-risk individuals has been monitored by regular testing of serumlevels of cholesterol in the various lipoprotein compartments.

[0005] One method for specific HDL cholesterol esting is based on theselective precipitation of non-HDL lipoproteins in serum by polyanioniccompounds, such as dextran sulfate, heparin, and phosphotungstate, inthe presence of a group-II cation, such as Mg²⁺, Mn²⁺, and Ca²⁺. Thespecificity and degree of precipitation are dependent on a variety offactors, including the type and concentration of the polyanion/metalagent. In general, the order of recipitation of serum cholesterolparticles, with increasing concentration of polyanion, is VLDL, LDL, andHDL. HDL usually remains soluble at concentrations of heparin or dextransulfate which completely precipitate lower density particles, althoughminor apoE species of HDL may be co-precipitated with lower densityparticles. By selective precipitation of lower density particles, HDLserum cholesterol levels can be determined.

[0006] In a typical lipid assay procedure, a small volume of blood isdrawn and centrifuged to produce a clear plasma or serum sample fluid.The sample fluid is then aliquoted into several assay tubes, fordetermination of (a) total serum cholesterol, (b) triglycerides, and (c)HDL cholesterol. The HDL sample is precipitated, as above, and the lowerdensity particles are removed by filtration or centrifugation prior tocholesterol detection. The samples are then reacted with an enzyme mixcontaining cholesterol esterase, cholesterol oxidase, peroxidase and adye which can be oxidized to a distinctly colored product in thepresence of H₂O₂. The tubes may be read spectrophotometrically, and thedesired total, HDL and LDL cholesterol values determined.

[0007] Despite the accuracy and reliability which can be achieved withthe liquid-phase cholesterol assay just described, the assay has anumber of limitations for use in widespread screening. First, the methoduses a venous blood sample, requiring a trained technician to draw andfractionate the blood sample, and aliquot the treated blood toindividual assay tubes. At least one of the sample tubes (for HDLdetermination) must be treated with a precipitating agent, and furtherprocessed to remove precipitated material. Although some of theseprocedures can be automated, analytical machines designed for thispurpose are expensive and not widely available outside of largehospitals.

[0008] Co-owned U.S. Pat. Nos. 5,213,964, 5,213,965, 5,316,196 and5,451,370, each of which is incorporated herein by reference, disclosemethods and assay devices which substantially overcome many of theabove-mentioned problems associated with liquid-assay procedures formeasuring serum cholesterol levels. In one embodiment, the device isdesigned for measuring the concentration of HDL-associated cholesterolin a blood sample also containing LDL and VLDL particles. The deviceincludes a sieving matrix capable of separating soluble and precipitatedlipoproteins as a fluid sample migrates through the matrix. A reservoirassociated with the matrix is designed to release a precipitating agent,for selectively precipitating LDL and VLDL, as fluid sample is drawninto and through the matrix. This allows HDL separation from theprecipitated lipoproteins, based on faster HDL migration through thesieving matrix. The fluid sample, thus depleted of non-HDL lipoproteins,then migrates to a test surface where it is assayed for cholesterol.

[0009] It was found that treatment of blood with reagents used inselectively precipitating non-HDL blood lipoproteins resulted in bindingof a proportion of the HDL present in the sample to non-coated glassfibers, and that such binding of HDL to the glass fibers duringfiltering or transport often resulted in spuriously low HDL cholesterolvalues. This problem was addressed, in co-owned U.S. Pat. No. 5,451,370,by coating the glass fibers in the matrix used for precipitation/sievingand transport of the filtered sample with a hydrophilic polymer orsilylating reagent.

[0010] In addition to the necessity for such coating to minimize HDLloss, the above-referenced devices also present the possibility ofcontamination of the flow transport path with the precipitatingreagents. Such reagents could interfere with other assay chemistrytaking place on other regions of the multi-assay device. The presentinvention addresses and overcomes these problems.

[0011] Further methods and devices for measuring HDL cholesterol inblood samples are disclosed in EP 0408223 and EP 0415298 (Rittersdorf etal.), which describe a continuous assay method carried out on a teststrip comprising the following steps and corresponding elements.

[0012] The blood sample is applied to a separation layer for separatingcellular blood constituents. Driven by capillary forces or gravity, thesample flows through a further carrier containing soluble precipitatingagents, which, after dissolving in the serum sample, precipitate non-HDLlipoproteins contained in the sample.

[0013] In a further carrier, the precipitated constituents, above, arefiltered from the serum sample to prevent their interference with laterHDL quantification. In the same carrier, the sample is transported to aposition adjacent the HDL-quantification carrier, and is stored untilthe HDL-quantification step is to be started. Finally, the sample istransferred to an HDL-quantification layer, where HDL cholesterol in theserum sample is quantified by an enzymatic reaction.

[0014] A disadvantage of this assay design, which can affect theaccuracy of HDL quantification, is that the carrier functioning as areservoir allows migration of the precipitated constituents into thesample, which interfere with HDL quantification. In addition, during thestorage of the serum sample, HDL can be trapped by adhering to thecarrier fibers, precipitating reagents can cause further undesiredreactions, and the carrier can become clogged by the drying serumsample.

[0015] U.S. Pat. No. 5,135,716 (Thakore) discloses additional devicesand methods for HDL quantification in a blood fluid sample. In thesedevices, the fluid sample flows continuously, though an unbroken path,from an inlet well to a carrier for HDL quantification. Accordingly, theability to control sample volume entering the HDL test carrier, and tocontrol environmental conditions for the HDL assay, is limited. Nor dothe devices provide for simultaneous assay of various analytes from asingle fluid sample.

[0016] It is therefore the object of the present invention to provide aHDL assay device and method which overcome the above-noted prior artdisadvantages.

SUMMARY OF THE INVENTION

[0017] In one aspect, the invention provides an assay device formeasuring serum cholesterol associated with high-density lipoproteins(HDL) in a blood fluid sample also containing low density lipoproteins(LDL) and/or very low density lipoproteins (VLDL), the devicecomprising:

[0018] a sample distribution matrix effective to distribute a bloodfluid sample from a sample application region within the matrix to oneor more sample collection regions within the matrix;

[0019] an HDL assay element, in which HDL concentration can be assayed,spaced apart from the sample distribution matrix,

[0020] a reagent pad, disposed between the HDL assay element and thesample distribution matrix, and spaced apart from the matrix, thereagent pad containing a reagent effective to selectively remove non-HDLlipoproteins from the fluid sample, and

[0021] mounting means effective (a) to maintain the device in asample-distribution position,

[0022] wherein the HDL assay element and reagent pad are spaced apartfrom the matrix, and (b) to transfer the device to a test position,whereby the HDL assay element is placed or maintained in contact withthe reagent pad, and the reagent pad is, concurrent with or subsequentto the contact, brought into contact with the matrix. In a preferredembodiment, the mounting means is further effective to (c) transfer thedevice from the test position to a position in which the HDL assayelement and reagent pad are spaced apart from the matrix.

[0023] Preferably, a lower surface of the HDL assay element is attachedto an upper surface of the reagent pad, so that the two are in permanentcontact.

[0024] In one embodiment, the device further comprises a cassette bodyto which the sample distribution matrix is attached, and a reaction barto which the HDL assay element is attached. The mounting means iseffective to attach the reaction bar to the cassette body, and to adjustthe relative positions of the reaction bar and cassette body between thesample-distribution position and the test position, as described above.Typically, the cassette body further comprises a well for containing theblood fluid sample and a sieving pad for removing cellular bloodcomponents from the blood fluid sample, in fluid communication with thesample application region, such that fluid passes from the well throughthe sieving pad and to the sample matrix.

[0025] The HDL assay element typically contains reagents which, in thepresence of HDL cholesterol, produce a change in the assay element whichcan be detected optically. For use in conducting simultaneous assays ofmultiple analytes on a single sample, the device preferably includesadditional assay elements attached to the reaction bar, such that theseelements are brought into contact with the sample collection regionswhen the device is transferred to the testing position. Preferably, thereaction bar is optically transparent, or it includes windows throughwhich the assay elements are visible.

[0026] In another embodiment, the HDL assay element comprises abiosensor. Preferably, the biosensor is effective to electrochemicallymeasure production of oxygen or hydrogen peroxide, which is in turndependent on HDL-associated cholesterol concentration within the samplefluid. Additional assay elements may also comprise biosensors, effectiveto electrochemically measure production of oxygen or hydrogen peroxide,which is in turn dependent on analyte concentration within the samplefluid.

[0027] In another aspect, the invention provides a related method ofmeasuring serum cholesterol associated with high-density lipoproteins(HDL) in a blood fluid sample also containing low density lipoproteins(LDL) and/or very low density lipoproteins (VLDL). The method includesthe following operations:

[0028] (a) contacting the sample with an absorptive sample distributionmatrix through which the sample is distributed to one or more samplecollection sites;

[0029] (b) bringing into contact with such a sample collection site, afirst surface of a reagent pad, to which sample fluid is transferred,containing a reagent effective to selectively remove non-HDLlipoproteins from the fluid,

[0030] wherein an opposite surface of the reagent pad is in simultaneouscontact with an HDL assay element, containing assay reagents effectiveto produce an indication of HDL cholesterol concentration, such thatsuccessive sample volumes proceed from the reagent pad to the HDL assayelement, and

[0031] (c) determining the level of HDL cholesterol in the sample bydetection at the HDL assay element.

[0032] The blood fluid sample is generally filtered, e.g. via a sievingmatrix upstream of the sample distribution matrix, to remove cellularblood components. Step (b) above comprises adjusting the device from asample-distribution position to a test position, as described above.Preferably, the method also includes the step of breaking the contactbetween the sample collection site and the first surface of the reagentpad, when a desired amount of sample fluid has been transferred. Fluidcontact is thus selectively establishable between the sample matrix andthe reagent pad which is connected to the HDL assay element. By theabove measures, effective sample distribution and volume control isrealized, and sample overload of the corresponding pads/test elements isprevented.

[0033] The reagent effective to selectively remove non-HDL lipoproteinsfrom the fluid sample, by binding or precipitation, may include, forexample, a sulfonated polysaccharide, such as dextran sulfate. In oneembodiment, the reagent pad is impregnated with the reagent in a formwhich is soluble in the fluid sample, and is of a material effective toentrap precipitated non-HDL lipoproteins within the reagent pad. Inanother embodiment, the reagent is immobilized to the reagent pad, andnon-HDL lipoproteins are removed from the sample by binding to theimmobilized reagent.

[0034] In a preferred embodiment, the reagent pad comprises a porouspolymeric membrane. The membrane may be an asymmetric polymericmembrane, having a smaller pored surface and an opposite, larger poredsurface. In this case, it is preferably oriented such that its smallerpored surface faces the HDL assay element.

[0035] The HDL assay element may also be a porous polymeric membrane,such as an asymmetric polymeric membrane, preferably oriented such thatits larger pored surface faces the reagent pad. In one embodiment, eachof the HDL assay element and the reagent pad is an asymmetric polymericmembrane, and the membranes are laminated such that the smaller poredsurface of the reagent pad contacts a larger pored surface of the HDLassay element.

[0036] In another embodiment, the HDL assay element and/or other assayelements comprise a biosensor, as described above.

[0037] The invention provides several advantages over the prior artdescribed above. For example, when the reagent pad containing theprecipitating or binding reagent contacts the sample fluid, it is indirect contact with the HDL assay element, thus limiting the temporalcontact of the blood sample with these reagents prior to the HDL assayreaction.

[0038] If desired, the test method can also be adapted to meet requiredenvironmental conditions. Accordingly, the assay can be stopped for adesired time after the sample application and removal of cellularcomponents, but prior to contact with binding or precipitation reagents,e.g. to adjust the surrounding atmosphere or adapt the environmentaltemperature to support the testing. This is accomplished by maintainingthe device in the sample-distribution position. To this end, the sampledistribution matrix is designed to additionally serve as a reservoir, ifneeded.

[0039] In the present method, the sample preparation and the HDLevaluation are carried out in separate steps. Sample preparationincludes, for example, filtering of cellular blood components and,optionally, temporary storage of the blood sample and adaptation of theblood sample to such test requirements or conditions as temperature,pressure and environmental atmosphere. The HDL evaluation step comprisesa time-effective removal of non-HDL constituents and a reliable HDLquantification. By this measure, the temporal contact of the bloodsample with the different reagents is reduced, and any chemicalinterference with the HDL evaluation is prevented. Further, the bloodsample is not extensively stored in a small pored carrier, since the HDLevaluation step is executed in a short period.

[0040] These and other objects and features of the invention will becomemore fully apparent when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is a side view of a multi-analyte assay device constructedin accordance with one embodiment of the invention;

[0042]FIG. 2 is a perspective view, in exploded form, of a multi-analyteassay device constructed in accordance with one embodiment of theinvention; and

[0043]FIG. 3 is a cross section view of two contacting asymmetricmembranes, for use as reagent pad and HDL assay element in oneembodiment of the invention, in a preferred orientation.

DETAILED DESCRIPTION OF THE INVENTION

[0044] I. Definitions

[0045] The terms below have the following meanings unless indicatedotherwise.

[0046] An element is in “fluid communication” with another element whena fluid is able to travel from one element to the other via capillaryaction and/or gravity. The elements may be in direct contact, but do notneed to be in direct contact; i.e., other elements through which saidfluid can pass may be intervening. An element is “not in fluidcommunication” with another element when a fluid is not able to travelfrom one element to the other via capillary action and/or gravity.Typically, the elements are physically separated, i.e. spaced apart.

[0047] A “pad”, such as a reagent pad or assay pad, as used herein, maycomprise any material, such as a porous membrane or fibrous strip, whichcan contain impregnated or immobilized reagents and through which fluidcan move via capillary action and/or gravity.

[0048] II. Assay Device

[0049]FIGS. 1 and 2 illustrate two embodiments of a multiple-analyteassay device 14 constructed in accordance with the present invention,with FIG. 2 shown in exploded format. The device is designedparticularly for determining serum cholesterol associated with HDL (alsoreferred to as HDL-associated cholesterol or simply HDL cholesterol)using a small volume of blood sample, typically between 10-50 μl ofblood. Other assays, such as total cholesterol or triglyceride level,can be determined simultaneously from the same sample. Determination ofHDL-associated cholesterol may also be referred to simply asdetermination of HDL or an HDL assay.

[0050] The apparatus includes a main body or support 15 which defines awell 16 dimensioned and sized to receive a quantity of a blood sample,typically between about 25-50 μl. The well is in fluid contact with asieving pad 22, which may be carried in a notched region 20 formed inthe upper edge of the support. The fluid contact may be direct, or as inthe device shown in FIG. 1, provided by a capillary conduit 18 formed inthe plate at the base of the well. The support is preferably a plasticplate, with the well, notched region and/or capillary formed by standardmolding or machining methods.

[0051] Sieving pad 22 carried in region 20 functions to partially removelarge particulate matter (including blood cells) as the sample migratesthrough the pad matrix in a bottom-to-top direction as shown in thefigure. Pad 22 is preferably formed of a glass fibrous matrix ofmaterial designed to draw aqueous fluid by surface wetting, and toretard the movement of blood cells as the blood sample is drawn throughthe matrix. That is, the pad serves as a chromatographic medium forseparating cell-size particles from soluble serum components on thebasis of different migration rates through the medium. One exemplary padis a glass fiber filter, such as a GF/D or PD008 filter supplied byWhatman, having a packing density of about 0.16 g/cm³. The pad is cut toside dimensions of about 3×8 mm, and a thickness of about 1 mm. The padis dimensioned to absorb a defined volume of sample fluid, preferablybetween about 15-25 μl. Sieving pad 22 may additionally contain redblood cell capture reagents, such as lectins, antibodies specific forred blood cell surface membrane proteins, thrombin, or ion exchangeagents.

[0052] The sieving pad, 22, in turn, contacts an elongate strip orsample distribution matrix 26 which extends along the upper edge ofplate 15. This strip may also be supported by foam cushions 27 or othersupports, as shown in FIG. 2. Matrix 26 serves to distribute samplefluid from a central sample-application region 28 of the strip, which isin fluid contact with pad 22, to opposite sample-collection regions 30,32 adjacent the ends of the matrix. The matrix is preferably formed ofglass fibers. The packing density and thickness of the matrix are suchas to absorb and distribute volumes of sample fluid, e.g., 10-25 μl,supplied to the sample-application region of the strip to thesample-collection regions of the strip. The matrix has a preferredpacking density between about 0.16 g/cm³ and 4.0 g/cm³. One exemplarystrip material is a F-165-25A glass fiber filter available from Whatman,having a packing density of about 0.2 gm/cm³ and a thickness of about0.12 mm.

[0053] Because the sample does not contact the glass fibers used for thesieving pad and sample distribution matrix in the presence ofprecipitation or binding reagent, coating as described in U.S. Pat. No.5,451,370, is not required to prevent adhesion of HDL. However, ifdesired, the glass fibers may be coated, e.g. with 5% polyvinyl alcoholby weight.

[0054] Device 14 also includes a reaction bar 60 composed of an elongatesupport 62, and multiple wettable assay elements 64, 66, 68 and 70,carried on the lower surface of the support, at the positions shown.Support 62 preferably is transparent or has windows, e.g. window 76(FIG. 2), which may simply be openings in the support, which allow thepads to be viewed through the support. The assay elements in thereaction bar can be attached to the support by a transparent ortranslucent adhesive material, or by sonic welding or other suitablebonding method. Each pad used in a particular assay containsanalyte-dependent reagents effective to produce an analyte-dependentchange in the pad which can be detected optically, either visually or bya detector, or via a biosensor, as described further below. All or anyintegral subset of the pads may be employed in a particular assay.

[0055] Desirably, the assay elements are porous polymer membranes,preferably having a thickness of about 100-150 μm and side dimensions ofabout 3 mm. The absorption volume of each element is preferably betweenabout 0.5-1.0 μl. In one embodiment, the assay elements, and inparticular that used for HDL assay, are asymmetric membranes; that is,membranes having a porosity gradient across the thickness of themembrane, as described further below. The assay elements may alsocomprise a biosensor, as described below.

[0056] The reaction bar is mounted on support 15 by mounting meanseffective to (a) maintain the device in a sample-distribution position,wherein the assay elements and reagent pads are spaced apart from thematrix, and to (b) transfer the device to a test position, where the HDLassay element is placed (or maintained, if the two pads are attachedtogether) in contact with the reagent pad, and the reagent pad is,concurrently or subsequently, brought into contact with matrix 26 at asample collection site. The mounting means can also be used to breaksuch contact after a desired amount of sample has entered the assayelements, and/or after a determined contact time, by transferring thedevice from the test position to a position in which the assay elementsand reagent pads are spaced apart from the matrix (which may be the sameas the “sample-distribution” position). Such transferring can becontrolled by monitoring the reflectance at the top surface of the assayelement, which reflects extent of wetting, as described in co-owned U.S.Pat. No. 5,114,350. Alternatively, when the absorption capacity and rateof sample uptake of the wettable materials are known, the quantity ofsample can be controlled with sufficient accuracy simply by using apredetermined contact time.

[0057] The mounting means can include, for example, a pair of resilientmembers, such as elastomeric blocks 71, 72, which act to bias the padstoward a non-transfer or sample-distribution position, at which the padsare spaced apart from the sample distribution matrix, with a spacingtypically of between about 0.5 to 1.0 mm. By compression or release ofthe resilient members, contact between sample distribution matrix 26 andreagent pad 74 and HDL assay element 64 can be selectively establishedand separated. The support blocks could be compressed by means ofsprings or a piston-like action. Alternatively, external mechanicaldevices could engage the main body 15 and/or support 62 and move onetowards the other. Such devices may include conventional components suchas clamps, pistons, stepper motors, worm gears, or the like. Anexemplary system is the Cholestech LDX® Analyzer, a self-contained,automated analyzer advantageous for use with assay devices such asdescribed herein.

[0058] In a preferred embodiment, at least one of the assay elements,used for assaying HDL, has affixed thereto a reagent pad 74, as shown inthe Figures. Alternatively, such a reagent pad 74 may be supported in asubstantially coplanar position between the HDL assay element, withwhich it may or may not be in contact, and a sample collection region ofmatrix 26. For example, a compressible support element could support thereagent pad above the matrix, such that movement of the reaction bartowards the main body (or vice versa) would first bring assay element 64into contact with the upper surface of reagent pad 74, and would thenbring the lower surface of the reagent pad into contact with the sampledistribution matrix. The reagent pad preferably has a thickness of about100-150 μm, side dimensions of about 3 mm, and an absorption volume ofabout 0.5-1.0 μl.

[0059] Reagent pad 74, preferably in contact with a selected assayelement used for HDL measurement, such as assay element 64 in thedrawings, contains a reagent used to selectively remove LDL and VLDLparticles from the fluid sample. Such reagents are known in the art asprecipitating reagents; see e.g. a review by P S Bachorik et al.,Methods in Enzymology 78-100 (1986). They include polyanionic compounds,such as sulfonated polysaccharides, heparin, or phosphotungstate, in thepresence of a group-II cation, such as Mg²⁺, Mn²⁺, and Ca²⁺. A preferredreagent is a sulfonated polysaccharide, such as dextran sulfate, havinga typical molecular weight of 50,000 to 500,000 daltons, in combinationwith magnesium acetate or chloride, buffered to maintain neutral pH.

[0060] The reagent pad is effective to entrap bound or precipitatednon-HDL lipoproteins within the reagent pad and prevent them fromentering HDL assay element 64. While a glass fiber filter can be usedfor such a purpose, such glass fibers should be coated to preventbinding HDL in the presence of the reagents, as described in U.S. Pat.No. 5,451,370, cited above. In a preferred embodiment, reagent pad 74 iscomposed of a porous polymeric membrane, as described further below.

[0061] The reagent pad contains reagents for selective removal ofnon-HDL lipoproteins, as described above. In one embodiment, a membraneis impregnated with such reagents. For example, a polysulfone asymmetricmembrane, as described below, is impregnated with an aqueous solutioncontaining dextran sulfate and a magnesium salt, such as magnesiumacetate, and dried. An exemplary procedure for preparing such membranesfor incorporation into the device is described in Example 1. In thiscase, the soluble precipitating reagents are released into the samplesolution as it penetrates the membrane.

[0062] In another embodiment, the reagents are immobilized to themembrane. Preferably, the negatively charged reagent, e.g. dextransulfate, is immobilized by electrostatic forces and/or covalently to amembrane having positively charged surface groups. An exemplary materialfor this purpose is a nylon membrane having surface quaternary ammoniumgroups, such as the AM080 membrane provided by Cuno Corp. (Meridian,Conn.).

[0063] In this case, the membrane acts as an affinity separation medium,such that non-HDL lipoproteins bind to the reagent affixed to themembrane, rather than precipitating, and are thereby separated from thesample fluid.

[0064] Other commercial polymeric membranes having a cationic surfaceinclude Immobilon-Ny+™ (Millipore Corp., Bedford, Mass.), Zetabind®(also from Cuno Corp.), GeneScreen® (NEN/DuPont, Boston, Mass.), HybondN+ (Amersham, Piscataway, N.J.) and Posidyne® (Pall Corp., Glen Cove,N.Y.). U.S. Pat. No. 5,543,054 (Charkoudian et al.) describes a methodfor covalently binding negatively charged carbohydrates to a membranehaving reactive moieties in proximity to positively charged moieties onits surface. The membrane is, for example, a porous polymer, e.g.polytetrafluroethylene, polyvinylidene fluoride, polyester, polyamide,polycarbonate, polypropylene, polymethylmethacrylate, polymethacrylate,polysulfone, or polystyrene, coated with Hercules R-4308™ apolyamido-polyamine epichlorohydrin resin

[0065] In one embodiment, reagent pad 74 is composed of an asymmetricmembrane; that is, a membrane having a pore size gradient across itsthickness. An asymmetric membrane is particularly preferred for use withprecipitating reagents incorporated into the membrane in soluble form,for optimum entrapment of precipitate. The preparation of asymmetricmembranes is described, for example, in U.S. Pat. Nos. 4,629,563,5,171,445, 5,886,059, 5,536,408, 5,562,826, and 4,774,192; in D. R.Lloyd, “Materials Science of Synthetic Membranes”, ACS Symposium 269:1-21 (1985). They are commercially available in a variety of pore sizesand pore size ratios. Materials of fabrication include polysulfones,polyethersulfones, polyamides, polyether amides, polyurethanes,cellulose acetate, polyvinyl pyrrolidone, polystyrenes and modifiedpolystyrenes, as well as blends, copolymers, and laminar composites. Anexemplary asymmetric membrane is a polysulfone or polyethersulfonemembrane, such as FILTERITE™ membranes provided by USF Filtration andSeparations (San Diego, Calif.). Minimum pore sizes typically range from0.01 to 1.0 μm, with maximum/minimum pore size ratios up to 100 or more.Thickness is typically 100-150 μm.

[0066] The asymmetric membrane is preferably oriented with its largerpored surface facing the sample application region; that is, facingdownward in FIGS. 1 and 2, and its smaller pored surface facing, andpreferably contacting, an assay element, e.g. assay element 64,containing reagents for assaying HDL level, as described further below.This orientation allows free access of sample into the pad through thelarger pores, and prevents passage of precipitated material, formed asthe solution contacts soluble precipitating agent, through the smallerpores, which are generally 1 μm or less in diameter. This pore size isalso preferred for non-asymmetric membranes.

[0067] In one embodiment, reagent pad 74 consists of a single membrane.The invention also contemplates the use of multiple stacked membranes,i.e. up to about six, where at least one and preferably each membranecontains reagents for binding or precipitation of non-HDL lipoproteins,for reagent pad 74. They may contain immobilized reagent, as describedabove, or they may be impregnated with soluble reagent. In the lattercase, asymmetric membranes are preferred, and are preferably orientedsuch that the smaller pored surface of the uppermost membrane facesassay element 64, and the larger pored surface of the lowest membranefaces the sample application region.

[0068] In one embodiment, assay element 64 is also a polymeric membrane,containing reagents for assaying HDL level, and may be an asymmetricmembrane as described above. In order to present the more uniformsurface for optical scanning and quantitation of assay results, anasymmetric membrane employed for assay element 64 is oriented with itssmaller pored surface facing upward, and its larger pored surface facingreagent pad 74.

[0069] Alternatively, an asymmetric membrane employed for assay element64 may be oriented with its larger pored surface facing upward and itssmaller pored surface facing reagent pad 74. This orientation is moresuitable for assays in which a visual, qualitative reading is to be madefrom the upper surface.

[0070] If desired, HDL assay reagents, such as peroxidase, may beimmobilized to the assay element membrane, according to well knownmethods for enzyme immobilization. (See e.g. U.S. Pat. No. 4,999,287;U.S. Pat. No. 5,419,902; Blum, L. J. et al., Anal. Lett. 20(2):317-26(1987); Kiang, S. W. et al., Clin. Chem. 22(8):1378-82.(1976);Guilbault, G. G., Ed., Modern Monographs in Analytical Chemistry, Vol.2: Analytical Uses of Immobilized Enzymes (1984); Torchilin, V. P.,Progress in Clinical Biochemistry and Medicine, Vol. 11: ImmobilizedEnzymes in Medicine (1991).) In another embodiment, a reagent, such ascatalase, which is effective to decompose any generated hydrogenperoxide that might diffuse downward from assay element 64, may beincluded in reagent pad 74.

[0071] In a preferred embodiment, where two attached polymeric membranesare employed for assay element 64 and reagent pad 74, respectively, theappropriate reagents are impregnated or immobilized, and the membranesare processed as a two-membrane layer for incorporation into the assaydevice during manufacture. An exemplary two-membrane layer comprisingtwo asymmetric membranes is shown in cross section in FIG. 3, with thepreferred orientation shown, with larger pores at 78 and smaller poresat 80.

[0072] In a further embodiment, the HDL assay element comprises abiosensor, as described, for example, in PCT Pubn. No. WO 9958966(Dobson et al.), which is incorporated herein by reference. Thisdocument discloses a microscale biosensor device, comprising aconducting surface, a layer of dielectric material overlying theconducting surface, and a plurality of pores extending through thedielectric layer. Each of the pores can act as a microelectrode,converting a chemical response into an electrical signal, by virtue of abiopolymer within the pore in contact with the conducting surface. Inuse, a fluid containing an analyte to be assayed is applied to the poresso as to be in contact with the biopolymer. In the present HDL assaydevice, this can be achieved by placing reagent pad 74 in fluid contactwith the HDL assay element; that is, the pore-containing surface of thebiosensor.

[0073] A counter electrode is provided which is in electrical contactwith the conducting surface via the sample fluid. A voltage is beapplied between the counter electrode and the conducting surface, andthe current that flows therebetween is measured. The measured current isindicative of the amount of a chosen analyte in the assayed fluid.

[0074] The microelectrodes preferably function as amperometricbiosensors. Briefly, an amperometric biosensor functions by theproduction of a current when a potential is applied between twoelectrodes. An example is the Clark oxygen electrode, which measurescurrent produced by reduction of oxygen or oxidation of hydrogenperoxide.

[0075] The dependence of such biosensors on dissolved oxygenconcentration can be overcome by the use of ‘mediators’, which transferthe electrons directly to the electrode, bypassing the reduction of theoxygen co-substrate. Ferrocenes represent a commonly used family ofmediators.

[0076] The biopolymer within the microelectrode pores is typically anenzyme, such as, for the measurement of HDL-associated cholesterol,cholesterol oxidase. Cholesterol is oxidized by cholesterol oxidase tothe corresponding ketone, liberating hydrogen peroxide, which can thenbe converted to water and oxygen by the enzyme peroxidase. Either oxygenor hydrogen peroxidase is then measured electrochemically at thebiosensor.

[0077] III. Assay Method

[0078] In operation, a blood sample is placed into well 16, and isimbibed by capillary action through sieving matrix 22, where largeparticulates, including red blood cells, are removed, and thence intosample distribution matrix 26. These steps take place while the deviceis in a “sample-distribution” position, such that the sampledistribution matrix does not contact the reagent or assay elements. Whenthe serum sample reaches the sample-collection sites, such as sites 30and 32 adjacent the ends of matrix 26, the device is adjusted to a testposition, preferably by moving reaction bar 60, to place assay elements66, 68, and 70 and reagent pad/assay element 74/64 (in the embodimentshown in the Figures) in contact with the matrix. In this position,sample fluid in the matrix is drawn into each contacted pad by capillaryflow, with fluid movement occurring in a direction normal to the padsurfaces. The plate is held at this position until a desired degree ofwetting of the pads is achieved. The plate is then moved, if desired, tobreak contact between the sample distribution matrix and the assayelements and reagent pad(s), when a desired amount of sample fluid hasentered the assay elements, and/or after an appropriate contact time,e.g. as described in Example 2 below.

[0079] In embodiments of the device in which reagent pad 74 is notaffixed to assay element 64, respective movement of the reaction bar andmain body toward each other, typically by moving the reaction bardownward, first places assay element 64 in contact with reagent pad 74,to approximate the arrangement of elements shown in the Figures, andfurther movement then places the reagent pad in contact with the sampledistribution matrix. Contact is maintained until a desired degree ofwetting is achieved, as described above.

[0080] Sample serum entering reagent pad 74 contacts precipitating orbinding reagent contained in the membrane, such that non-HDLlipoproteins are selectively precipitated and retained by filtration, inthe case of soluble reagent, or bound to the membrane, in the case ofimmobilized reagent. The membrane is thus effective to entrap non-HDLlipoproteins, while allowing passage of serum containing liquid-phaseHDL to HDL assay element 64. The HDL assay element contains reagents forquantification of HDL-associated cholesterol. Preferably, these includecholesterol esterase, for releasing free cholesterol from HDL;cholesterol oxidase, for producing H₂O₂ by reaction with freecholesterol; peroxidase, which converts H₂O₂ to oxygen and water; and acoupled dye system which is converted, in the presence of peroxidase andH₂O₂, to a distinctively colored signal reaction product. Alternatively,the generated oxygen or H₂O₂ may be measured by the use of a biosensor,as described above.

[0081] During operation, as sample fluid passes through the HDL assaypath, comprising pads 74 and 64, its leading edge passes in an upwarddirection through pad 74, where non-HDL lipoproteins react and areentrapped, and directly to adjacent assay element 64, where HDL reactswith the assay reagents therein, for measurement of HDL-associatedcholesterol. Further portions of sample continue to be in contact withpad 74 during this time, and proceed from pad 74 to pad 64 in a likemanner, until the absorption capacity is reached. Accordingly,quantification of HDL-associated cholesterol in assay element 64 occursconcurrently with the precipitation or binding reaction taking place inreagent pad 74. Preferably, the volume of sample fluid transferred tothe HDL assay path (comprising elements 74 and 64) from the sampledistribution matrix is equal to or greater than the absorption capacityof assay element 64, and less than or equal to the combined absorptioncapacity of assay element 64 and reagent pad 74.

[0082] One advantage of the current device and method is that the sampledistribution path does not contain non-HDL precipitating or bindingreagents; such reagents are present only in reagent pad 74. Therefore,the possibility of interference from these reagents, in assays ofanalytes other than HDL, is eliminated.

[0083] Preferably, each of the assay elements contains reagentcomponents for producing H₂O₂ via reaction of the analyte with anenzyme; the H₂O₂ subsequently converts a substrate reagent to a coloredsignal reaction product, or is measured electrochemically, as describedabove. Such components include, for example, peroxidase and a coupleddye system which is converted by the peroxidase, in the presence ofH₂O₂, to a distinctively colored signal reaction product. Enzymaticcolor reactions which employ a variety of substrate-specific oxidases,for enzymatic generation of H₂O₂, and subsequent oxidation of a dye toform a colored reaction product, are well known.

[0084] A device having four or more reaction pads can be used tosimultaneously measure HDL cholesterol (HDL), glucose, total cholesterol(TCh), and triglyceride lipid (TG). Each pad contains theabove-described common pathway components (peroxidase and a coupled dyesystem) such that generated H₂O₂ can be measured or produces adistinctly colored signal reaction product. The total cholesterol assayelement, which is exposed to serum without exposure to a precipitatingor binding reagent, and the HDL assay elements each include, in additionto the common pathway components, cholesterol esterase, for releasingesterified cholesterol in free-cholesterol form from serum lipoproteins,including HDL, LDL, and VLDL particles, and cholesterol oxidase, forproducing H₂O₂ by reaction with free cholesterol in the sample fluid, asdescribed above. The glucose assay pad includes glucose oxidase, inaddition to the common-pathway components. The triglyceride padincludes, in addition to the common-pathway components, lipase,L-glycerol kinase, and L-glycerol-3-phosphate oxidase, for generatingH₂O₂ from triglyceride, via the intermediate L-glycerol-3-phosphate. Theserum sample drawn into the TG pad is not exposed to precipitating orbinding reagents, and thus contains all of the serum lipoproteins, sothe TG signal represents total serum triglycerides.

[0085] Reference standard pads may also be employed; see, for example,the system described in co-owned U.S. Pat. No. 5,114,350, which isincorporated herein by reference.

[0086] As noted above, one advantage of the current device and method isthat the sample distribution matrix does not contain non-HDLprecipitating or binding reagents; such reagents are present only inreagent pad 74. Therefore, the possibility of interference by thesereagents, in assays of analytes such as total serum cholesterol andtotal triglycerides, is eliminated.

EXAMPLES

[0087] The following examples are provided to illustrate but not tolimit the invention.

Example 1

[0088] Preparation of Reagent Membrane with Soluble Precipitant and HDLTest Membrane

[0089] To prepare a reagent membrane with soluble precipitant, anaqueous solution containing 1 mg/ml dextran sulfate (500,000 MW) and12.5 mM Mg(OAc)₂ is dispensed onto a polysulfone asymmetric membrane0.22 inches in width. The membrane thickness is 127+/−5 μm, with abubble point of 85+/−5 psi. The reagent is dispensed at a rate of 16.6ul/inch, and the membrane is dried for 20 minutes at 50° C. in acontinuous roll process. Lengths of e.g. 100 feet are prepared in thismanner and cut to fit the assay devices.

[0090] To prepare an HDL reaction membrane, a similar asymmetricpolysulfone membrane is impregnated with the following aqueousformulation: cholesterol oxidase 36.5 Units/ml, cholesterol esterase 215Units/ml, peroxidase 200 Units/ml, 4-aminoantipyrine 1.88 μm/ml, andTOOS (3-[ethyl(3-methylphenyl)amino]-2-hydroxy propanesulfonic acid)12.05 μm/ml. Dispense rate and drying time are as for the reagentmembrane, above.

[0091] The two membranes may be attached separately (sequentially) tothe reaction bar by ultrasonic welding, or they may be attachedsimultaneously with a single ultrasonic weld step.

Example 2

[0092] Assay Procedure

[0093] The following assays were carried out in an LDX® analyzer, usingreagent membranes and HDL assay elements prepared essentially asdescribed in Example 1. Sample (35 μl of serum or whole blood) wasapplied to the sample well and allowed to distribute through the sampledistribution matrix for 2 minutes. The reaction bar was then contactedwith the matrix for 3 seconds, a time sufficient to transfer enoughserum to fill the reagent pad and assay element (combined capacity about1.5 μl), after which the bar was returned to its original position.Reflectance readings were taken from the upper surface of the HDL assayelement every 3 seconds for 150 seconds, to monitor the progress of theHDL assay reaction. The minimum reflectance value attained was thenconverted to mg/dL of HDL cholesterol according to a previouslyestablished calibration curve.

[0094] The concentration values below (mg/dL) are from five serumsamples analyzed as described above and on a Beckman reference analyzer,showing excellent correlation. Sample No. HDL assay Beckman referenceA010904 23.8 24.5 10906 43.0 41.9 10502 61.5 59.6 10801 80.6 78.7 1080592.4 87.2

It is claimed:
 1. An assay device for measuring serum cholesterolassociated with high-density lipoproteins (HDL) in a blood fluid samplealso containing low density lipoproteins (LDL) or very low densitylipoproteins (VLDL), the device comprising: a sample distribution matrixeffective to distribute a blood fluid sample from a sample applicationregion within the matrix to one or more sample collection regions withinthe matrix; an HDL assay element, in which HDL concentration can beassayed, spaced apart from said sample distribution matrix, a reagentpad, disposed between said HDL assay element and said sampledistribution matrix, and spaced apart from said matrix, said reagent padcontaining a reagent effective to selectively remove non-HDLlipoproteins from the fluid sample, and mounting means effective (a) tomaintain said device in a sample-distribution position, wherein said HDLassay element and reagent pad are spaced apart from said matrix, and (b)to transfer said device to a test position, whereby the HDL assayelement is placed or maintained in contact with the reagent pad, and thereagent pad is, concurrent with or subsequent to said contact, broughtinto contact with said matrix.
 2. The device of claim 1, wherein a lowersurface of the HDL assay element is attached to an upper surface of thereagent pad.
 3. The device of claim 1, further comprising a cassettebody to which said sample distribution matrix is attached.
 4. The deviceof claim 3, further comprising a reaction bar to which said HDL assayelement is attached.
 5. The device of claim 4, wherein said mountingmeans is effective to attach said reaction bar to said cassette body andto adjust the relative positions of the reaction bar and cassette bodybetween said sample-distribution position and said test position.
 6. Thedevice of claim 3, wherein said cassette body further comprises a wellfor containing said blood fluid sample and a sieving pad for removingcellular blood components from said blood fluid sample, in fluidcommunication with said sample application region.
 7. The device ofclaim 4, further comprising additional assay elements attached to saidreaction bar, such that said pads are brought into contact with saidsample collection regions when the device is transferred to the testingposition.
 8. The device of claim 1, wherein said mounting means isfurther effective to (c) transfer the device from said test position toa position in which said HDL assay element and reagent pad are spacedapart from said matrix.
 9. The device of claim 1, wherein said reactionbar is optically transparent or includes windows through which saidassay elements are visible.
 10. The device of claim 1, wherein saidreagent includes a sulfonated polysaccharide.
 11. The device of claim 1,wherein said reagent pad is impregnated with said reagent in a formwhich is soluble in said fluid sample, and is of a material effective toentrap precipitated non-HDL lipoproteins within the reagent pad.
 12. Thedevice of claim 1, wherein said reagent is immobilized to said reagentpad.
 13. The device of claim 1, wherein said HDL assay element containsreagents which, in the presence of HDL-associated cholesterol, produce achange in the assay element which can be detected optically.
 14. Thedevice of claim 1, wherein said HDL assay element comprises a biosensor.15. The device of claim 14, wherein said biosensor is effective tomeasure production of oxygen or hydrogen peroxide which is dependent onHDL-associated cholesterol concentration within said element.
 16. Thedevice of claim 1, wherein said reagent pad comprises a porous polymericmembrane.
 17. The device of claim 15, wherein said reagent pad is anasymmetric polymeric membrane, having a smaller pored surface and anopposite, larger pored surface.
 18. The device of claim 17, wherein saidmembrane is oriented such that its smaller pored surface faces the HDLassay element.
 19. The device of claim 1, wherein each of said HDL assayelement and said reagent pad is an asymmetric polymeric membrane, andsaid membranes are laminated such that the smaller pored surface of thereagent pad contacts a larger pored surface of the HDL assay element.20. A method of measuring serum cholesterol associated with high-densitylipoproteins (HDL) in a blood fluid sample also containing low densitylipoproteins (LDL) or very low density lipoproteins (VLDL), comprising(a) contacting the sample with an absorptive sample distribution matrixthrough which said sample is distributed to one or more samplecollection sites; (b) bringing into contact with such a samplecollection site, a first surface of a reagent pad, to which said sampleis transferred, containing a reagent effective to selectively removenon-HDL lipoproteins from the fluid sample, wherein an opposite surfaceof said reagent pad is in simultaneous contact with an HDL assayelement, containing assay reagents effective to produce an indication ofHDL cholesterol concentration, such that successive sample volumesproceed from said reagent pad to said HDL assay element, and (c)determining the level of HDL cholesterol in said sample by detection atsaid HDL assay element.
 21. The method of claim 20, further comprisingthe step of breaking said contact between the sample collection site andthe first surface of the reagent pad, when a desired amount of samplehas been transferred.
 22. The method of claim 20, wherein said reagentpad is impregnated with said reagent in a form which is soluble in saidfluid sample, and is of a material effective to entrap precipitatednon-HDL lipoproteins within the reagent pad.
 23. The method of claim 20,wherein said reagent effective to selectively remove non-HDLlipoproteins is immobilized to said reagent pad.
 24. The method of claim22, wherein said reagent pad comprises an asymmetric polymeric membrane,having a smaller pored surface and an opposite, larger pored surface.25. The method of claim 24, wherein said membrane is oriented such thatits smaller pored surface faces said HDL assay element.
 26. The methodof claim 20, wherein said HDL assay element comprises a biosensor. 27.The method of claim 26, wherein said biosensor is effective to measureproduction of oxygen or hydrogen peroxide which is dependent onHDL-associated cholesterol concentration within said element.
 28. Themethod of claim 24, wherein each of said HDL assay element and saidreagent pad is an asymmetric polymeric membrane, and said membranes arelaminated such that the smaller pored surface of the reagent padcontacts a larger pored surface of the HDL assay element.